Unraveling the roles of sphingolipids in plant innate immunity.
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ABSTRACT: The first step in the biosynthesis of sphingolipids is the condensation of serine and palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5'-phosphate (PLP)-dependent alpha-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits, both of which are bound to the endoplasmic reticulum (ER) most likely with the type I topology, whereas other members of the POAS family are soluble homodimer enzymes. LCB2 appears to be unstable unless it is associated with LCB1. Potent inhibitors of SPT structurally resemble an intermediate in a probable multistep reaction mechanism for SPT. Although SPT is a housekeeping enzyme, its activity is regulated transcriptionally and post-transcriptionally, and its up-regulation is suggested to play a role in apoptosis induced by certain types of stress. Specific missense mutations in the human LCB1 gene cause hereditary sensory neuropathy type I, an autosomal dominantly inherited disease, and these mutations confer dominant-negative effects on SPT activity.Biochimica et Biophysica Acta 07/2003; 1632(1-3):16-30. · 4.66 Impact Factor
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ABSTRACT: Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 resistant 11-1 (fbr 11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B(1) (FB(1)), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr 11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB(1). By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1-phosphate in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.Cell Research 01/2008; 17(12):1030-40. · 10.53 Impact Factor
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ABSTRACT: Fumonisins (FB) and AAL-toxin are sphingoid-like compounds produced by several species of fungi associated with plant diseases. In animal cells, both fumonisins produced by Fusarium moniliforme and AAL-toxin produced by Alternaria alternata f. sp. lycopersici inhibit ceramide synthesis, an early biochemical event in the animal diseases associated with consumption of F. moniliforme-contaminated corn. In duckweed (Lemna pausicostata Heglem. 6746), tomato plants (Lycopersicon esculentum Mill), and tobacco callus (Nicotiana tabacum cv Wisconsin), pure FB1 or AAL-toxin caused a marked elevation of phytosphingosine and sphinganine, sphingoid bases normally present in low concentrations. The relative increases were quite different in the three plant systems. Nonetheless, disruption of sphingolipid metabolism was clearly a common feature in plants exposed to FB1 or AAL-toxin. Resistant varieties of tomato (Asc/Asc) were much less sensitive to toxin-induced increases in free sphinganine. Because free sphingoid bases are precursors to plant "ceramides," their accumulation suggests that the primary biochemical lesion is inhibition of de novo ceramide synthesis and reacylation of free sphingoid bases. Thus, in plants the disease symptoms associated with A. alternata and F. moniliforme infection may be due to disruption of sphingolipid metabolism.Plant physiology 12/1994; 106(3):1085-1093. · 6.56 Impact Factor
[Plant Signaling & Behavior 4:6, 536-538; June 2009]; ©2009 Landes Bioscience
536 Plant Signaling & Behavior2009; Vol. 4 Issue 6
It has long been known that fungal pathogens like Fusarium
and Alternaria spp. produce toxins (mycotoxin) to kill plant
cells. These mycotoxins have been shown to perturb the plant
sphingolipid biosynthesis pathway, resulting in the necrotic cell
death of plant cells. A recent study by Shi et al.1 revealed that an
increase in the amount of cellular sphingoid bases triggers plant
programmed cell death (PCD) through accumulation of reactive
oxygen species (ROS). These findings point to the importance of
sphingolipids in the regulation of plant cell in disease develop-
ment as well as in defense responses. In the latest report,2 we
showed that serine palmitoyltransferase (SPT), the key enzyme
of sphingolipid biosynthesis, regulates not only plant cell death
but also defense response against a non-host pathogen, soliciting
further studies to elucidate the roles of sphingolipids in plant
Sphingolipids are defined as lipids containing sphingoid bases
(1,3-dihydroxy-2-amino-alkane and its derivatives) as a structural
backbone, and are essential components of membranes in all
eukaryotes. The sphingolipids and their metabolites in yeast and
animals are known to be powerful generators of a variety of signals
involved not only in maintaining cellular homeostasis but also in
stress responses and apoptosis (reviewed in refs. 3 and 4). Moreover,
they are key components of the membrane microdomains called
lipid rafts, playing important roles in cellular processes including
signal transduction, membrane trafficking, cytoskeletal organiza-
tion and pathogen entry.5
Sphingolipid biosynthesis is initiated by a decarboxyl condensa-
tion of serine with palmitoyl-CoA, a reaction catalyzed by serine
palmitoyltransferase (SPT) to form 3-ketosphinganine in a pyri-
doxal 5’-phosphate (PLP) dependent manner. 3-ketosphinganine
is immediately converted to sphinganine (dihydrosphingosine)
Unraveling the roles of sphingolipids in plant innate immunity
Yoshihiro Takahashi,1,2 Thomas Berberich,1 Hiroyuki Kanzaki,1 Hideo Matsumura,1 Hiromasa Saitoh,1 Tomonobu
Kusano2 and Ryohei Terauchi1,3,*
1Iwate Biotechnology Research Center; Kitakami, Iwate Japan; 2Graduate School of Life Sciences; Tohoku University; Aoba; Sendai, Miyagi Japan; 3Graduate School of
Agricultural Sciences; Iwate University; Morioka, Iwate Japan
Key words: cell death, defense response, serine palmitoyltransferase, sphingolipid, plant
Figure 1. Sphingolipid biosynthesis pathway in plant. Inhibition steps by
myriocin, Fumonisin B1 and AAL-toxin are indicated.
*Correspondence to: Ryohei Terauchi; Iwate Biotechnology Research Center;
Division of Genetics and Genomics; 22-174-4; Narita; Kitakami, Iwate 024-0003
Japan; Tel.: +22.214.171.12411; Fax: +126.96.36.19981; Email: terauchi@ibrc.
Submitted: 09/15/08; Accepted: 03/31/09
Previously published online as a Plant Signaling & Behavior E-publication:
Addendum to: Takahashi Y, Berberich T, Kanzaki H, Matsumura H, Saitoh H,
Kusano T, et al. Serine palmitoyltransferase, the first step enzyme in sphingolipid
biosynthesis, is involved in non-host resistance. Mol Plant-Microbe Interact 2009;
22:31–8; PMID: 19061400; DOI: 10.1094/MPMI-22-1-0031.
www.landesbioscience.comPlant Signaling & Behavior 537
Unraveling the roles of sphingolipids in plant innate immunity
domain is necessary for NbLCB2 to cause cell death. To test the
involvement of NbLCB2 in plant resistance against pathogens, we
analyzed NbLCB2 transcription following inoculation of plants
with the non-host bacterial pathogen Pseudomonas cichorii and
the host pathogen Pseudomonas syringae pv. tabaci. During the
early stages of infection, NbLCB2 mRNA is strongly induced by
P. cichorii, but not by P. syringae pv. tabaci, suggesting the involve-
ment of SPT in non-host resistance. To confirm this finding, we
measured pathogen growth in plants whereby SPT function was
compromised by the SPT inhibitor myriocin. Resistance of N.
benthamiana against P. cichorii was compromised by myriocin
treatment, whereas the one against a host pathogen P. syringae pv.
tabaci was not affected. Moreover, silencing of NbLCB2 as well as
NbLCB1 genes compromised non-host resistance of N. bentha-
miana against P. cichorii. The combined results suggest that de
novo sphingolipid biosynthesis is important for innate immunity
against non-host pathogen. Whether this reduction in plant non-
host resistance is a consequence of reduction in HR-like cell death
or caused by impairment of an as yet unidentified defense signaling
pathway remains to be determined.
The details of sphingolipid biosynthesis regulation and the indi-
vidual function of these molecules are still largely uncharacterized.
However, the recent novel findings strongly suggest that sphingo-
lipids play a crucial role in plant innate immunity including cell
death. The manipulation of host signaling and the co-option of
cell death pathways by plant pathogens by means of their effector
molecules (such as AAL toxin or fumonisin) is a fascinating and
exciting area of study.19
This work was carried out in part by support from “Program for
Promotion of Basic Research Activities for Innovative Biosciences”
(Japan), “Iwate University 21st Century COE Program:
Establishment of Thermo-Biosystem Research Program” and
MAFF Japan (Genomics for Agricultural Innovation PMI-0010)
to R.T., and by “The Sumitomo Foundation” to Y.T. Thanks are
due to Matt Shenton and Muluneh Tamiru Oli for improving the
1. Shi L, Bielawski J, Mu J, Dong H, Teng C, Zhang J, et al. Involvement of sphingoid
bases in mediating reactive oxygen intermediate production and programmed cell death
in Arabidopsis. Cell Res 2007; 17:1030-40.
2. Takahashi Y, Berberich T, Kanzaki H, Matsumura H, Saitoh H, Kusano T, et al. Serine
palmitoyltransferase, the first step enzyme in sphingolipid biosynthesis, is involved in
non-host resistance. Mol Plant-Microbe Interact 2009; In press.
3. Hannun YA, Obeid LM. Principles of bioactive lipid signalling: lessons from sphingolip-
ids. Nat Rev Mol Cell Biol 2008; 9:139-50.
4. Morales A, Lee H, Goñi FM, Kolesnick R, Fernandez-Checa JC. Sphingolipids and cell
death. Apoptosis 2007; 12:923-39.
5. Munro S. Lipid rafts: Elusive or illusive? Cell 2003; 115:377-88.
6. Hanada K. Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism.
Biochim Biophys Acta 2003; 1632:16-30.
7. Gable K, Slife H, Bacikova D, Monaghan E, Dunn TM. Tsc3p is an 80-amino acid pro-
tein associated with serine palmitoyltransferase and required for optimal enzyme activity.
J Biol Chem 2000; 275:7597-603.
8. Yasuda S, Nishijima M, Hanada K. Localization, topology and function of the LCB1
subunit of serine palmitoyltransferase in mammalian cells. J Biol Chem 2003; 278:4176-
9. Chen M, Han G, Dietrich CR, Dunn TM, Cahoon EB. The essential nature of sphin-
golipids in plants as revealed by the functional identification and characterization of the
Arabidopsis LCB1 subunit of serine palmitoyltransferase. Plant Cell 2006; 18:3576-93.
by a 3-ketosphinganine reductase, and sphinganine is converted
to dihydroceramide by sphinganine N-acyltransferase (acyl-CoA-
dependent ceramide synthase) (Fig. 1). SPT is suggested to be
the key enzyme for the regulation of sphingolipid levels in cells.6
In yeast and mammals, SPT is a heterodimer consisting of LCB1
and LCB2 subunits. The PLP binding motif (T[FL][GTS]K[SAG]
[FLV]G) around the PLP-binding lysine residue is present in
LCB2, but not in LCB1.6 Recently, it has been reported that
the LCB1 subunit is involved in the stabilization of the LCB2
protein in yeast and mammalian cells.7,8 Both subunits and the
PLP-binding motif in LCB2 are also conserved in plants.9
Several lines of evidence implicate the roles of sphingolipids
in plant cell death regulation. Necrotrophic fungi Fusarium
moniliforme and Alternaria alternata f sp. lycopersici produce
sphinganine-analogous mycotoxins, fumonisin B1 and AAL-toxin,
respectively, to kill the host plant via disruption of sphin-
golipid metabolism resulting from inhibition of sphinganine
N-acyltransferase activity (Fig. 1).10-12 AAL-toxin-induced cell
death is associated with the accumulation of 3-ketosphinganine
and sphinganine in susceptible tomato leaves. This cell death
process is blocked by the SPT inhibitor, myriocin, suggesting
that the increase in the cellular sphingolipid imetermediates
is crucial for the observed cell death.11 The Arabidopsis lesion
mimic mutants accelerated cell death (acd) 5 and acd11 exhibiting
spontaneous cell death phenotypes were shown to have defects in
the genes encoding a putative ceramide kinase and a sphingosine
transfer protein, respectively, also pointing to the involvement of
sphingolipid intermediates in plant cell death.13,14 Recently Shi
et al.1 identified a fumonisin B1-resistant Arabidopsis mutant,
fbr11-1, that fails to initiate cell death upon fumonisin
B1-treatment. The authors showed that fbr11-1 had a defect in
the AtLCB1 gene. Taken together, these observations suggest that
an excess of cellular sphingolipids intermediates, more specifically
of 3-ketosphinganine and sphinganine, results in plant cell death.
Shi et al.1 tested this hypothesis by directly applying free sphingoid
bases such as sphinganine, phytosphingosine and sphingosine to
plant cells, and showed that the treatment indeed caused reactive
oxygen species (ROS) production followed by programmed cell
death (PCD)-like cell death. They also suggested that the balance
between phosphorylated vs. free (un-phosphorylated) sphingoid
bases may determine the fate of plant cell for death or survival.
Our recent study by Takahashi et al.2 supports the previous
observations, and further indicated the involvement of sphingo-
lipids in non-host resistance. We performed a high-throughput
screening of cell death-inducing factors in plants.15,16 A cDNA
library from Nicotiana benthamiana leaves was cloned into a
binary potato virus X (PVX)-based expression vector and trans-
formed into Agrobacterium. Individual Agrobacterium colonies
were inoculated into N. benthamiana leaves by toothpicks. After
1 to 2 weeks of inoculation, cell death was observed around
the inoculated site, if the protein expressed from the integrated
cDNA has cell death-inducing activity in plant cells. Using this
system, we identified various novel genes from N. benthamiana as
cell death-inducing factors,17,18 one of which encoded the LCB2
subunit of SPT (NbLCB2).2 A deletion study indicated that PLP
Unraveling the roles of sphingolipids in plant innate immunity
538Plant Signaling & Behavior2009; Vol. 4 Issue 6
10. Abbas H, Tanaka T, Duke SO, Poter JK, Wray EM, Hodges L, et al. Fumonisin- and
AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free
sphingoid bases. Plant Physiol 1994; 106:1085-93.
11. Spassieva SD, Markham JE, Hille J. The plant disease resistance gene Asc-1 prevents
disruption of sphingolipid metabolism during AAL-toxin-induced programmed cell
death. Plant J 2002; 32:561-72.
12. Wang H, Li J, Bostock RM, Gilchrist DG. Apoptosis: a functional paradigm for pro-
grammed cell death induced by a host-selective phytotoxin and invoked during develop-
ment. Plant Cell 1996; 8:375-91.
13. Brodersen P, Petersen M, Pike HM, Olszak B, Skov S, Ødum N, et al. Knockout of
Arabidopsis ACCELERATED-CELL-DEATH11 encoding a sphingosine transfer prtein
causes activation of programmed cell death and defense. Gene Dev 2002; 16:490-502.
14. Liang H, Yao N, Song JT, Luo S, Lu H, Greenberg JT. Ceramides modulate programmed
cell death in plants. Gene Dev 2003; 17:2636-41.
15. Berberich T, Takahashi Y, Saitoh H, Terauchi R. High-throughput functional screening
of genes in planta. In: Kahl G, Meksem K, eds. The Handbook of Plant Functional
Genomics. Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA 2008; 113-36.
16. Terauchi R, Nasir KHB, Ito A, Saitoh H, Berberich T, Takahashi Y. High-throughput
functional screening of plant and pathogen genes in planta. Plant Biotech 2005; 22:455-
17. Coemans B, Takahashi Y, Berberich T, Ito A, Kanzaki H, Matsumura H, et al. High-
throughput in planta expression screening identifies an ADP-ribosylation factor (ARF1)
involved in non-host resistance and R gene-mediated resistance. Mol Plant Pathol 2008;
18. Nasir KHB, Takahashi Y, Ito A, Saitoh H, Matsumura H, Kanzaki H, et al. High-throughput
in planta expression screening identifies a class II ethylene-responsive element binding
factor-like protein that regulates plant cell death and non-host resistance. Plant J 2005;
19. Hogenhout SA, van der Hoorn RAL, Terauchi R, Kamoun S. Emerging concepts in
effector biology of plant-associated organisms. Mol Plant-Microbe Interact 2009; In